Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION Transient activation cogen synthase kinase-3␤ (GSK-3␤; for review, see Peifer ␤ and Polakis 2000). This complex promotes phosphoryla- of -catenin signaling tion of ␤-catenin at a number of N-terminal serine and in cutaneous keratinocytes is threonine residues, and the phosphorylated ␤-catenin is ubiquitinated and subsequently degraded by the protea- sufficient to trigger the active some. growth phase of the hair cycle Binding of Wnts to their cognate frizzled and low-den- sity lipoprotein receptor-related protein receptor com- in mice plexes on the cell surface leads to inhibition of GSK-3␤ activity and increased levels of free ␤-catenin in the cell David Van Mater,1 Frank T. Kolligs,2,6 (Peifer and Polakis 2000). In cancers, inactivating muta- Andrzej A. Dlugosz,3,5,7 and Eric R. Fearon1,2,4,5,8 tions in the APC or axin proteins or activating mutations affecting N-terminal phosphorylation sites in ␤-catenin 1Departments of Human Genetics, 2Internal Medicine, lead to stabilization of ␤-catenin (Polakis 2000). Regard- 3Dermatology, and 4Pathology, and the 5Cancer Center, less of whether Wnt signals or mutational defects stabi- University of Michigan School of Medicine, lize ␤-catenin, following its accumulation in the cell, Ann Arbor, Michigan 48109, USA ␤-catenin can complex in the nucleus with T cell factor/ lymphoid enhancer factor (TCF/LEF) transcription regu- Wnts have key roles in many developmental processes, lators, leading to activation of TCF-regulated genes (a list including hair follicle growth and differentiation. Stabi- of candidate TCF target genes is provided at: http:// lization of ␤-catenin is essential in the canonical Wnt www.stanford.edu/∼rnusse/wntwindow.html). signaling pathway. We developed transgenic mice ex- Wnt/␤-catenin signaling has been proposed to function pressing a regulated form of ␤-catenin in the skin. in hair follicle morphogenesis and differentiation (Kishi- Chronic activation of ␤-catenin in resting (telogen) hair moto et al. 2000; Fuchs et al. 2001; Millar 2002). Hair follicles resulted in changes consistent with induction of follicle morphogenesis is characterized by the downward growth of epithelial hair follicle precursor cells into the an exaggerated, aberrant growth phase (anagen). Tran- ␤ embryonic dermis, where follicle precursor cells envelop sient activation of -catenin produced a normal anagen. the mesenchymal dermal papilla to produce the bulb re- Our data lend strong support to the notion that a Wnt/ gion of the follicle. Cells in the outermost epithelial cell ␤ -catenin signal operating on hair follicle precursor cells compartment, the outer root sheath (ORS), continue to serves as a crucial proximal signal for the telogen–anagen proliferate and migrate downward to the bulb region transition. throughout the active growth phase of the hair follicle. At the same time, rapidly-proliferating matrix cells in Received January 17, 2003; revised version accepted April 2, the bulb of mature follicles migrate upward in the cen- 2003. tral portion of the follicle to give rise to six distinct cell types in the maturing hair shaft and surrounding inner Wnt family proteins function in short-range signaling. root sheath (IRS). Wnt signaling appears to be involved in They regulate cell fate, adhesion, differentiation, prolif- patterning in the skin, as mice with a skin-specific de- ␤ eration, and motility, and many studies have demon- letion of -catenin (Huelsken et al. 2001) or mice with strated their critical roles in development (for review, see constitutive overexpression of an inhibitor of Wnt sig- Peifer and Polakis 2000). Mutational defects in Wnt sig- naling in skin (Andl et al. 2002) fail to develop hair fol- naling have a major contributing role in a broad spec- licles. Likewise, Lef-1 knockout mice are characterized trum of cancers (Polakis 2000). At present, it appears by arrested development of hair follicles and a complete many Wnts exert their effects, at least in part, through a lack of hair (van Genderen et al. 1994). Conversely, mice ␤ “canonical” signaling pathway in which stabilization of expressing a constitutively activated form of -catenin the ␤-catenin protein is essential. Much of the ␤-catenin lacking critical phosphorylation sites in the N terminus ⌬ ␤ protein in the cell is associated with the cell membrane, ( N87 -catenin) in their skin display evidence of de where ␤-catenin binds to and links E-cadherin to the novo hair follicle morphogenesis in the interfollicular cytoskeleton through ␤-catenin’s binding to ␣-catenin. A epithelium as they age (Gat et al. 1998). These studies ␤ fraction of the ␤-catenin is free in the cytoplasm and/or demonstrate a role for Wnt/ -catenin signaling during nucleus. Normally, in the absence of Wnt signals, initial development of hair follicles. ␤-catenin is bound and negatively regulated by a protein Once established, hair follicles undergo a growth cycle complex that includes the adenomatous polyposis coli consisting of periods of growth (anagen), regression (cata- (APC) and axin tumor suppressor proteins as well as gly- gen), and rest (telogen; Muller-Rover et al. 2001). The bulge activation theory proposes the dermal papillae ac- tivate anagen by signaling the stem cell compartment of [Keywords: ␤-catenin; hair follicle; transgenic mouse; anagen; hair cycle] the hair follicle residing in the bulge (for review, see 6Present address: Medizinische Klinik II, Klinikum Grosshadern, Uni- Stenn and Paus 2001). There is evidence consistent with versity of Munich, 81377 Munich, Germany. Wnt/␤-catenin signaling having a role in anagen induc- Corresponding authors. tion. For example, an increase in ␤-galactosidase is seen 7E-MAIL [email protected]; FAX (734) 763-4575. 8E-MAIL [email protected]; FAX (734) 647-7979. in the bulge region of the hair follicle at the onset of Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ anagen in TOPgal transgenic mice carrying a TCF/LEF gad.1076103. optimal promoter upstream of a ␤-galactosidase gene GENES & DEVELOPMENT 17:1219–1224 © 2003 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/03 $5.00; www.genesdev.org 1219 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Van Mater et al. (DasGupta and Fuchs 1999). Also, the first postnatal ana- gen does not occur in mice in which ␤-catenin expres- sion is progressively lost in the skin (Huelsken et al. 2001). Finally, Wnts 10a and 10b are expressed in post- natal hair follicles at anagen onset, but not in resting follicles (Reddy et al. 2001). These data are consistent with the view that a Wnt signal activates ␤-catenin sig- naling in the bulge, thereby driving the resting follicle into active growth. Whereas the data imply a role for ␤-catenin in the telo- gen–anagen transition, no studies have examined the ef- fect of inducible and reversible ␤-catenin signaling in the skin, with the goal of modeling the transient activation resulting from effects of canonical Wnt signaling. There- fore, we sought to develop a system in which we could tightly regulate ␤-catenin function in the mouse skin. We have shown previously the function of a chimeric ␤-catenin protein, in which a full-length ␤-catenin poly- peptide with a codon 33 activating mutation (serine-to- tyrosine substitution—S33Y) is fused in-frame to a mu- tated version of the hormone binding domain of mouse estrogen receptor-␣ (ER), is rapidly activated by the li- gand 4-hydroxytamoxifen (4-OHT) in cultured cells (Kol- ligs et al. 2002). We expressed the S33Y␤-catenin–ER fu- sion protein in the skin of transgenic mice using the Figure 1. Activation of the K5/S33Y␤-catenin–ER protein by well-characterized bovine keratin 5 (K5) promoter 4-OHT and expression in transgenic mice. (A) Schematic of the K5/ (Ramirez et al. 1994). We explored effects of chronic and S33Y␤-catenin–ER construct. A cDNA encoding the chimeric pro- transient activation of ␤-catenin signaling in cutaneous tein was subcloned into a bovine K5 expression cassette. (B) Activity keratinoyctes by topical application of 4-OHT. We show of the K5/S33Y␤-catenin–ER protein is inducible by 4-OHT in 1811 here that prolonged activation of ␤-catenin signaling re- keratinocytes. The 1811 keratinocytes were transfected with an ␤ sults in profound proliferation of the ORS and other epi- empty K5 cassette, the pBabe/S33Y -catenin–ER construct, or the K5/S33Y␤-catenin–ER construct along with either the TCF-respon- thelial components of the hair follicle. Transient signal- sive reporter construct TOPFLASH or control FOPFLASH construct. ing results in activation of a normal anagen phase. These Cells were then treated with either 4-OHT in ethanol, or ethanol data demonstrate ␤-catenin signaling provides a potent alone, and harvested 30 h later to assess luciferase activity. The growth stimulus for hair follicle progenitor cells and is assays were performed in duplicate; data are reported as the ratio of sufficient, when transiently activated in epithelial hair relative light units for TOPFLASH:FOPFLASH, normalized for transfection efficiency. (C) Expression of the K5/S33Y␤-catenin–ER follicle precursors, to trigger telogen to anagen transi- fusion protein relative to endogenous ␤-catenin in transgenic mouse tion. lines. Protein was isolated from tail skin of F1 mice derived from three independently derived founders and subjected to Western